The Na(+)-K(+)-ATPase alpha2-subunit isoform modulates contractility in the perinatal mouse diaphragm

This study uses genetically altered mice to examine the contribution of the Na⁺-K⁺-ATPase ₂ catalytic subunit to resting potential, excitability, and contractility of the perinatal diaphragm. The ₂ protein is reduced by 38% in ₂-heterozygous and absent in ₂-knockout mice, and ₁-isoform is upregulated 1.9-fold in ₂-knockout. Resting potentials are depolarized by 0.8–4.0 mV in heterozygous and knockout mice. Action potential threshold, overshoot, and duration are normal. Spontaneous firing, a developmental function, is impaired in knockout diaphragm, but this does not compromise its ability to fire evoked action potential trains, the dominant mode of activation near birth. Maximum tetanic force, rate of activation, force-frequency and force-voltage relationships, and onset and magnitude of fatigue are not changed. The major phenotypic consequence of reduced ₂ content is that relaxation from contraction is 1.7-fold faster. This finding reveals a distinct cellular role of the ₂-isoform at a step after membrane excitation, which cannot be restored simply by increasing ₁ content. Na⁺/Ca²⁺ exchanger expression decreases in parallel with ₂-isoform, suggesting that Ca²⁺ extrusion is affected by the altered ₂ genotype. There are no major compensatory changes in expression of sarcoplasmic reticulum Ca²⁺-ATPase, phospholamban, or plasma membrane Ca²⁺-ATPase. These results demonstrate that the Na⁺-K⁺-ATPase ₁-isoform alone is able to maintain equilibrium K⁺ and Na⁺ gradients and to substitute for ₂-isoform in most cellular functions related to excitability and force. They further indicate that the ₂-isoform contributes significantly less at rest than expected from its proportional content but can modulate contractility during muscle contraction. Na⁺-K⁺-ATPase ₂ catalytic subunit; heterozygous mice; knockout mice; resting potential     

Insulin increases plasma membrane content and reduces phosphorylation of Na+-K+ pump alpha 1-subunit in HEK-293 cells

Insulin stimulates K⁺ uptake andNa⁺ efflux via the Na⁺-K⁺ pump inkidney, skeletal muscle, and brain. The mechanism of insulin action inthese tissues differs, in part, because of differences in the isoformcomplement of the catalytic -subunit of theNa⁺-K⁺ pump. To analyze specifically the effectof insulin on the ₁-isoform of the pump, we have studiedhuman embryonic kidney (HEK)-293 cells stably transfected with the ratNa⁺-K⁺ pump ₁-isoform tagged onits first exofacial loop with a hemagglutinin (HA) epitope. The plasmamembrane content of ₁-subunits was quantitated bybinding a specific HA antibody to intact cells. Insulin rapidly increased the number of ₁-subunits at the cell surface.This gain was sensitive to the phosphatidylinositol (PI) 3-kinaseinhibitor wortmannin and to the protein kinase C (PKC) inhibitorbisindolylmaleimide. Furthermore, the insulin-stimulated gain insurface -subunits correlated with an increase in the binding of anantibody that recognizes only the nonphosphorylated form of₁ (at serine-18). These results suggest that insulinregulates the Na⁺-K⁺ pump in HEK-293 cells, atleast in part, by decreasing serine phosphorylation and increasingplasma membrane content of ₁-subunits via a signalingpathway involving PI 3-kinase and PKC.

     

The Na+ pump and intracellular signaling mechanisms

The main properties of Na+ /K(+)-ATPase as a natural receptor for cardiotonic steroids have been discusses. Primary attention is focused on structural and functional differences between the alpha-subunit isoforms of Na+/K(+)-ATPase in different tissues. General information on the role of the Na pump in signaling cascades in kidney epithelial cells, cardiomyocytes and neurons is presented. The data obtained indicate that, in neurons, several alpha-isoforms of Na+/K(+)-ATPase possessing different sensitivity to ouabain may have different signaling functions.     

Role of Na+/Ca2+ exchange and the plasma membrane Ca2+ pump in hormone-mediated Ca2+ efflux from pancreatic acini

Summary The relative contributions of the Na⁺/Ca²⁺ exchange and the plasma membrane Ca²⁺ pump to active Ca²⁺ efflux from stimulated rat pancreatic acini were studied. Na⁺ gradients across the plasma membrane were manipulated by loading the cells with Na⁺ or suspending the cells in Na⁺-free media. The rates of Ca²⁺ efflux were estimated from measurements of [Ca²⁺] _i using the Ca²⁺-sensitive fluorescent dye Fura 2 and⁴⁵Ca efflux. During the first 3 min of cell stimulation, the pattern of Ca²⁺ efflux is described by a single exponential function under control, Na⁺-loaded, and Na⁺-depleted conditions. Manipulation of Na⁺ gradients had no effect on the hormone-induced increase in [Ca²⁺] _i . The results indicate that Ca²⁺ efflux from stimulated pancreatic acinar cells is mediated by the plasma membrane Ca²⁺ pump. The effects of several cations, which were used to substitute for Na⁺, on cellular activity were also studied. Choline⁺ and tetramethylammonium⁺ (TMA⁺) released Ca²⁺ from intracellular stores of pancreatic acinar, gastric parietal and peptic cells. These cations also stimulated enzyme and acid secretion from the cells. All effects of these cations were blocked by atropine. Measurements of cholecystokinin-octapeptide (CCK-OP)-stimulated amylase release from pancreatic acini, suspended in Na⁺, TMA⁺, choline⁺, or N-methyl-d-glucamine⁺ (NMG⁺) media containing atropine, were used to evaluate the effect of the cations on cellular function. NMG⁺, choline⁺, and TMA⁺ inhibited amylase release by 55, 40 and 14%, respectively. NMG⁺ also increased the Ca²⁺ permeability of the plasma membrane. Thus, to study Na⁺ dependency of cellular function, TMA⁺ is the preferred cation to substitute for Na⁺. The stimulatory effect of TMA⁺ can be blocked by atropine.     

The membrane potential modulates the ATP-dependent Ca2+ pump of cardiac sarcolemma

The effect of membrane potential on the activity of the ATP-dependent Ca2+ pump of isolated canine ventricular sarcolemmal vesicles was investigated. The membrane potential was controlled by the intravesicular and extravesicular concentration of K+, and the initial rates of Ca2+ uptake both in the presence and the absence of valinomycin were determined. The rate of Ca2+ uptake was stimulated by a inside-negative potential induced in the presence of valinomycin. The valinomycin-dependent stimulation was enhanced by the addition of K+ channel blocker, tetraethylammonium ion or Ba2+. The electrogenicity of cardiac sarcolemmal ATP-dependent Ca2+ pump is suggested from the increase of Ca2+ uptake by negative potential induced by valinomycin.     

Intracellular H+ regulates the alpha -subunit of ENaC, the epithelial Na+ channel

Protons regulateelectrogenic sodium absorption in a variety of epithelia, including thecortical collecting duct, frog skin, and urinary bladder. Recently,three subunits (, , ) coding for the epithelial sodium channel(ENaC) were cloned. However, it is not known whether pH regulatesNa⁺ channels directly byinteracting with one of the three ENaC subunits or indirectly byinteracting with a regulatory protein. As a first step to identifyingthe molecular mechanisms of proton-mediated regulation of apicalmembrane Na⁺ permeability inepithelia, we examined the effect of pH on the biophysical propertiesof ENaC. To this end, we expressed various combinations of -, -,and -subunits of ENaC in Xenopusoocytes and studied ENaC currents by the two-electrode voltage-clampand patch-clamp techniques. In addition, the effect of pH on the-ENaC subunit was examined in planar lipid bilayers. We report that ,,-ENaC currents were regulated by changes in intracellular pH(pH_i) but not by changes inextracellular pH (pH_o).Acidification reduced and alkalization increased channel activity by avoltage-independent mechanism. Moreover, a reduction ofpH_i reduced single-channel openprobability, reduced single-channel open time, and increased single-channel closed time without altering single-channel conductance. Acidification of the cytoplasmic solution also inhibited ,-ENaC, ,-ENaC, and -ENaC currents. We conclude thatpH_i but notpH_o regulates ENaC and that the-ENaC subunit is regulated directly bypH_i.     

Plasmalemmal Na+/Ca2+ exchanger modulates Ca2+-dependent exocytotic release of glutamate from rat cortical astrocytes

Astroglial excitability operates through increases in Ca²⁺_cyt (cytosolic Ca²⁺), which can lead to glutamatergic gliotransmission. In parallel fluctuations in astrocytic Na⁺_cyt (cytosolic Na⁺) control metabolic neuronal-glial signalling, most notably through stimulation of lactate production, which on release from astrocytes can be taken up and utilized by nearby neurons, a process referred to as lactate shuttle. Both gliotransmission and lactate shuttle play a role in modulation of synaptic transmission and plasticity. Consequently, we studied the role of the PMCA (plasma membrane Ca²⁺-ATPase), NCX (plasma membrane Na⁺/Ca²⁺ exchanger) and NKA (Na⁺/K⁺-ATPase) in complex and coordinated regulation of Ca²⁺_cyt and Na⁺_cyt in astrocytes at rest and upon mechanical stimulation. Our data support the notion that NKA and PMCA are the major Na⁺ and Ca²⁺ extruders in resting astrocytes. Surprisingly, the blockade of NKA or PMCA appeared less important during times of Ca²⁺ and Na⁺ cytosolic loads caused by mechanical stimulation. Unexpectedly, NCX in reverse mode appeared as a major contributor to overall Ca²⁺ and Na⁺ homoeostasis in astrocytes both at rest and when these glial cells were mechanically stimulated. In addition, NCX facilitated mechanically induced Ca²⁺-dependent exocytotic release of glutamate from astrocytes. These findings help better understanding of astrocyte-neuron bidirectional signalling at the tripartite synapse and/or microvasculature. We propose that NCX operating in reverse mode could be involved in fast and spatially localized Ca²⁺-dependent gliotransmission, that would operate in parallel to a slower and more widely distributed gliotransmission pathway that requires metabotropically controlled Ca²⁺ release from the ER (endoplasmic reticulum).     

Inositol trisphosphate-induced Ca2+ signaling modulates auxin transport and PIN polarity

The phytohormone auxin is an important determinant of plant development. Directional auxin flow within tissues depends on polar localization of PIN auxin transporters. To explore regulation of PIN-mediated auxin transport, we screened for suppressors of PIN1 overexpression (supo) and identified an inositol polyphosphate 1-phosphatase mutant (supo1), with elevated inositol trisphosphate (InsP(3)) and cytosolic Ca(2+) levels. Pharmacological and genetic increases in InsP(3) or Ca(2+) levels also suppressed the PIN1 gain-of-function phenotypes and caused defects in basal PIN localization, auxin transport and auxin-mediated development. In contrast, the reductions in InsP(3) levels and Ca(2+) signaling antagonized the effects of the supo1 mutation and disrupted preferentially apical PIN localization. InsP(3) and Ca(2+) are evolutionarily conserved second messengers involved in various cellular functions, particularly stress responses. Our findings implicate them as modifiers of cell polarity and polar auxin transport, and highlight a potential integration point through which Ca(2+) signaling-related stimuli could influence auxin-mediated development.     

Inhibition of Na(+)-independent H+ pump by Na(+)-induced changes in cell Ca2+

Apical membrane H+ extrusion in the renal outer medullary collecting duct, inner stripe, is mediated by a Na(+)-independent H+ pump. To examine the regulation of this transporter, cell pH and cell Ca2+ were measured microfluorometrically in in vitro perfused tubules using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and fura-2, respectively. Apical membrane H+ pump activity, assayed as cell pH recovery from a series of acid loads (NH3/NH+4 prepulse) in the total absence of ambient Na+, initially occurred at a slow rate (0.06 +/- 0.02 pH units/min), which was not sufficient to account for physiologic rates of H+ extrusion. Over 15-20 min after the initial acid load, the rate of Na(+)-independent cell pH recovery increased to 0.63 +/- 0.09 pH units/min, associated with a steady-state cell pH greater than the initial pre-acid load cell pH. This pattern suggested an initial suppression followed by a delayed activation of the apical membrane H+ pump. Replacement of peritubular Na+ with choline or N-methyl-D-glucosamine resulted in an initial spike increase in cell Ca2+ followed by a sustained increase in cell Ca2+. The initial rate of Na(+)-independent cell pH recovery could be increased by elimination of the Na+ removal-induced sustained cell Ca2+ elevation by: (a) performing studies in the presence of 135 mM peritubular Na+ (1 mM peritubular amiloride used to inhibit basolateral membrane Na+/H+ antiport); (b) clamping cell Ca2+ low with dimethyl-BAPTA, an intracellular Ca2+ chelating agent; or (c) removal of extracellular Ca2+. Cell acidification induced a spike increase in cell Ca2+. The late acceleration of Na(+)-independent cell pH recovery was independent of Na+ removal and of the method used to acidify the cell, but was eliminated by prevention of the cell Ca2+ spike and markedly delayed by the microfilament-disrupting agent, cytochalasin B. This study demonstrates that peritubular Na+ removal results in a sustained elevation in cell Ca2+, which inhibits the apical membrane H+ pump. In addition, rapid cell acidification associated with a spike increase in cell Ca2+ leads to a delayed activation of the H+ pump. Thus, cell Ca2+ per se, or a Ca(2+)-activated pathway, can modulate H+ pump activity.     

Integrin alpha(3)-subunit expression modulates alveolar epithelial cell monolayer formation

We investigated expression of the alpha(3)-integrin subunit by rat alveolar epithelial cells (AECs) grown in primary culture as well as the effects of monoclonal antibodies with blocking activity against the alpha(3)-integrin subunit on AEC monolayer formation. alpha(3)-Integrin subunit mRNA and protein were detectable in AECs on day 1 and increased with time in culture. alpha(3)- and beta(1)-integrin subunits coprecipitated in immunoprecipitation experiments with alpha(3)- and beta(1)-subunit-specific antibodies, consistent with their association as the alpha(3)beta(1)-integrin receptor at the cell membrane. Treatment with blocking anti-alpha(3) monoclonal antibody from day 0 delayed development of transepithelial resistance, reduced transepithelial resistance through day 5 compared with that in untreated AECs, and resulted in large subconfluent patches in monolayers viewed by scanning electron microscopy on day 3. These data indicate that alpha(3)- and beta(1)-integrin subunits are expressed in AEC monolayers where they form the heterodimeric alpha(3)beta(1)-integrin receptor at the cell membrane. Blockade of the alpha(3)-integrin subunit inhibits formation of confluent AEC monolayers. We conclude that the alpha(3)-integrin subunit modulates formation of AEC monolayers by virtue of the key role of the alpha(3)beta(1)-integrin receptor in AEC adhesion.     

Reduced volume but increased training intensity elevates muscle Na+-K+ pump alpha1-subunit and NHE1 expression as well as short-term work capacity in humans

The present study examined muscle adaptations and alterations in work capacity in endurance-trained runners after a change from endurance to sprint training. Fifteen runners were assigned to either a sprint training (ST, n = 8) or a control (CON, n = 7) group. ST replaced their normal training by 30-s sprint runs three to four times a week, whereas CON continued the endurance training (approximately 45 km/wk). After the 4-wk sprint period, the expression of the muscle Na+-K+ pump alpha1-subunit and Na+/H+-exchanger isoform 1 was 29 and 30% higher (P < 0.05), respectively. Furthermore, plasma K+ concentration was reduced (P < 0.05) during repeated intense running. In ST, performance in a 30-s sprint test, Yo-Yo intermittent recovery test, and two supramaximal exhaustive runs was improved (P < 0.05) by 7, 19, 27, and 19%, respectively, after the sprint training period, whereas pulmonary maximum oxygen uptake and 10-k time were unchanged. No changes in CON were observed. The present data suggest a role of the Na+-K+ pump in the control of K+ homeostasis and in the development of fatigue during repeated high-intensity exercise. Furthermore, performance during intense exercise can be improved and endurance performance maintained even with a reduction in training volume if the intensity of training is very high.     

Phospholemman modulates Na+/Ca2+ exchange in adult rat cardiac myocytes

Previous studies have shown that overexpression of phospholemman (PLM) affected contractile function and Ca(2+) homeostasis in adult rat myocytes. We tested the hypothesis that PLM modulated Na(+)/Ca(2+) exchanger (NCX1) activity. PLM was overexpressed in adult rat myocytes by adenovirus-mediated gene transfer. After 72 h, the half-time of relaxation from caffeine-induced contracture, an estimate of forward NCX1 activity, was prolonged 1.8-fold (P < 0.003) in myocytes overexpressing PLM compared with control myocytes overexpressing green fluorescent protein alone. Reverse NCX1 current (3 Na(+) out:1 Ca(2+) in) was significantly (P < 0.0001) lower in PLM myocytes, especially at more positive voltages. Immunofluorescence demonstrated colocalization of PLM and NCX1 to the plasma membrane and t-tubules. Resting membrane potential, action potential amplitude and duration, myocyte size, and NCX1 and calsequestrin protein levels were not affected by PLM overexpression. At 5 mM extracellular [Ca(2+)] ([Ca(2+)](o)), the depressed contraction amplitudes in PLM myocytes were increased towards normal by cooverexpression with NCX1. At 0.6 mM [Ca(2+)](o), the supranormal contraction amplitudes in PLM myocytes were reduced by cooverexpression with NCX1. We conclude that PLM modulated myocyte contractility partly by inhibiting Na(+)/Ca(2+) exchange.     

Na(+) entry via store-operated channels modulates Ca(2+) signaling in arterial myocytes

In manynonexcitable cells, hormones and neurotransmitters activateNa⁺ influx and mobilizeCa²⁺ from intracellular stores.The stores are replenished by Ca²⁺influx via "store-operated"Ca²⁺ channels (SOC). The mainroutes of Na⁺ entry in these cellsare unresolved, and no role forNa⁺ in signaling has beenrecognized. We demonstrate that the SOC are a majorNa⁺ entry route in arterialmyocytes. Unloading of the Ca²⁺stores with cyclopiazonic acid (a sarcoplasmic reticulumCa²⁺ pump inhibitor) and caffeineinduces a large externalNa⁺-dependent rise in thecytosolic Na⁺ concentration. Onecomponent of this rise in cytosolicNa⁺ concentration is likely due toNa⁺/Ca²⁺exchange; it depends on elevation of cytosolicCa²⁺ and is insensitive to 10 mMMg²⁺ and 10 µMLa³⁺. Another component isinhibited by Mg²⁺ andLa³⁺, blockers of SOC; thiscomponent persists in cells preloaded with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraaceticacid to buffer Ca²⁺ transients andpreventNa⁺/Ca²⁺exchange-mediated Na⁺ entry. ThisNa⁺ entry apparently is mediatedby SOC. The Na⁺ entry influencesNa⁺ pump activity andNa⁺/Ca²⁺exchange and has unexpectedly large effects on cell-wideCa²⁺ signaling. The SOC pathwaymay be a general mechanism by which Na⁺ participates in signaling inmany types of cells.

     

Regulation of SERCA Ca2+ pump expression by cytoplasmic Ca2+ in vascular smooth muscle cells

Vascular smooth muscle cells (VSMC) express three isoforms of the sarcoplasmic or endoplasmic reticulum Ca2+-ATPase (SERCA) pump; SERCA2b predominates (91%), whereas SERCA2a (6%) and SERCA3 (3%) are present in much smaller amounts. Treatment with thapsigargin (Tg) or A-23187 increased the level of mRNA encoding SERCA2b four- to fivefold; SERCA3 increased about 10-fold, whereas SERCA2a was unchanged. Ca2+ chelation prevented the Tg-induced SERCA2b increase, whereas Ca2+ elevation itself increased SERCA2b expression. These responses were discordant with those of 78-kDa glucose-regulated protein/immunoglobulin-binding protein (grp78/BiP), an endoplasmic reticulum stress-response protein. SERCA2b mRNA elevation was much larger than could be accounted for by the observed increase in message stability. The induction of SERCA2b by Tg did not require protein synthesis, nor was it affected by inhibitors of calcineurin, protein kinase C, Ca2+/calmodulin-dependent protein kinase, or tyrosine protein kinases. Treatment with the nonselective protein kinase inhibitor H-7 prevented Tg-induced SERCA2b expression from occurring, whereas another nonselective inhibitor, staurosporine, was without effect. We conclude that changes in cytosolic Ca2+ control the expression of SERCA2b in VSMC via a mechanism involving a currently uncharacterized, H-7-sensitive but staurosporine-insensitive, protein kinase.     

Double-pulse facilitation of smooth muscle alpha 1-subunit Ca2+ channels expressed in CHO cells.

Frequent strong depolarizations facilitate Ca2+ channels in various cell types by shifting their gating behavior towards mode 2, which is characterized by long openings and high probability of being open. In cardiac cells, the same type of gating behavior is potentiated by beta-adrenoceptors presumably acting via phosphorylation of a protein identical to or associated with the channel. Voltage-dependent phosphorylation has also been reported to underlie Ca2+ channel facilitation in chromaffin adrenal medulla and in skeletal muscle cells. We studied a possible voltage-dependent facilitation of the principal channel forming alpha 1-subunit of the dihydropyridine-sensitive smooth muscle Ca2+ channel. Single channel and whole-cell Ca2+ currents were recorded in Chinese hamster ovary cells stably expressing the class Cb Ca2+ channel alpha 1-subunit. Strong depolarizing voltage-clamp steps preceding the test pulse resulted in a 2- to 3-fold increase of the single Ca2+ channel activity and induction of mode 2-like gating behavior. Accordingly we observed a significant potentiation of the whole-cell current by approximately 50%. In contrast to the previous suggestions we found no experimental evidence for involvement of channel phosphorylation by protein kinases (cAMP-dependent protein kinase, protein kinase C and other protein kinases utilizing ATP gamma S) in the control and facilitated current. The data demonstrate that the L-type Ca2+ channel alpha 1-subunit solely expressed in Chinese hamster ovary cells is subject to a voltage-dependent facilitation but not to phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Na+/Ca2+ Exchange and Cellular Ca2+ Homeostasis

The Na⁺/Ca²⁺ exchange system is the primary Ca²⁺ efflux mechanism in cardiac myocytes, and plays an important role in controlling the force of cardiac contraction. The exchanger protein contains 11 transmembrane segments plus a large hydrophilic domain between the 5th and 6th transmembrane segments; the transmembrane regions are reponsible for mediating ion translocation while the hydrophilic domain is responsible for regulation of activity. Exchange activity is regulated in vitro by interconversions between an active state and either of two inactive states. High concentrations of cytosolic Na⁺ or the absence of cytosolic Ca²⁺ promote the formation of the inactive states; phosphatidylinositol-(4,5)bisphosphate (or other negatively charged phospholipids) and cytosolic Ca²⁺ counteract the inactivation process. The importance of these mechanisms in regulating exchange activity under normal physiological conditions is uncertain. Exchanger function is also dependent upon cytoskeletal interactions, and the exchanger's location with respect to intracellular Ca²⁺-sequestering organelles. An understanding of the exchanger's function in normal cell physiology will require more detailed information on the proximity of the exchanger and other Ca²⁺-transporting proteins, their interactions with the cytoskeleton, and local concentrations of anionic phospholipids and transported ions.     

Genetic manipulation of cardiac Na+/Ca2+ exchange expression

The Na+/Ca2+ exchanger (NCX) is the primary Ca2+ extrusion mechanism in cardiomyocytes. To further investigate the role of NCX in excitation-contraction coupling and Ca2+ homeostasis, we created murine models with altered expression levels of NCX. Homozygous overexpression of NCX resulted in mild cardiac hypertrophy. Decline of the Ca2+ transient and relaxation of contraction were increased and the reverse mode of NCX was augmented. Overexpression also led to a higher susceptibility to ischemia-reperfusion injury and to a greater ability of NCX to trigger Ca2+-induced Ca2+ release. Furthermore, an increase in peak L-type Ca2+ current was observed suggesting a direct influence of NCX on L-type Ca2+ current. Whereas global knockout of NCX led to prenatal death, a recently generated cardiac-specific NCX knockout mouse was viable with surprisingly normal contractile properties. Expression levels of other Ca2+-handling proteins were not altered. Ca2+ influx in these animals is limited by a decrease of peak L-type Ca2+ current. An alternative Ca2+ efflux mechanism, presumably the plasma membrane Ca2+-ATPase, is sufficient to maintain Ca2+-homeostasis in the NCX knockout mice.     

Cyclic stretch translocates the alpha2-subunit of the Na pump to plasma membrane in skeletal muscle cells in vitro

The Na+-K+-ATPase and its regulation is important for maintaining membrane potential and transmembrane Na(+) gradient in all skeletal muscle cells and thus is essential for cell survival and function. In our previous study, cyclic stretch activated the Na pump in cultured skeletal muscle cells. Presently, we investigated whether this stimulation was the result of translocation of Na+-K+-ATPase from endosomes to the plasma membrane, and also evaluated the role of phosphatidylinositol 3-kinase (PI 3-kinase), the activation of which initiated vesicular trafficking and targeting of proteins to specific cell compartments. Skeletal muscle cells were stretched at 25% elongation continuous for 24h using the Flexercell Strain Unit. The plasma membrane and endosome fractions were isolated and Western blotted to localize the Na+-K+-ATPase alpha1- and alpha2-subunit protein. The results showed stretch increased Na+-K+-ATPase alpha1- and alpha2-subunit protein expression in plasma membrane fractions and decreased it in endosomes. The alpha2-subunit had a more dynamic response to mechanical stretch. PI 3-kinase inhibitors (LY294002) blocked the stretch-induced translocation of the Na+-K+-ATPase alpha2-subunit, while LY294002 had no effect on the transfer of alpha1-subunit. We concluded that cyclic stretch mainly stimulated the translocation of the alpha2-subunit of Na+-K+-ATPase from endosomes to the plasma membrane via a PI 3-kinase-dependent mechanism in cultured skeletal muscle cells in vitro, which in turn increased the activity of the Na pump.